Project Summary/Abstract This proposal addresses the need to improve candidate vaccines for enterotoxigenic Escherichia coli (ETEC), one of the commonest causes of severe bacterial diarrhea worldwide. The disease primarily afflicts children in the poorest regions of the world, contributing to excessive morbidity and mortality associated with diarrheal illness. Vaccines targeting ETEC appear feasible based on protection observed in challenge models and the presumptive development of immunity to natural infection. Traditional vaccine approaches have been unable accommodate the extensive diversity of ETEC pathogens leading to my mentors discovery of novel antigens, involved in ETEC pathogenesis, that compliment traditional approaches. The main objective of this study is to advance the development of novel antigens as key contributors to an effective ETEC vaccine dovetailing with my continued career development. The two novel antigens are EatA, a mucinase degrading intestinal mucins allowing pathogen access to the host and EtpA, which binds to blood group A sugars on the intestinal epithelia. To advance my translational research career and pursue additional experience in human subjects research, I will advance the pre-clinical evaluation of these novel antigens to determine their utility in vaccine design and subsequent efficacy evaluation in future clinical trials. We will assess the conservation of novel ETEC antigens, EtpA and EatA through a variety of means including advanced sequencing and genomics analysis combined with functional analysis. The protection afforded by these antigens may differ based on blood type, impacting the interpretation of vaccine efficacy which will be assessed through retrospective, cross-sectional, and cohort studies. To achieve these aims, I will cultivate an ability to design, implement, and analyze data from human trials. I have assembled collaborators and mentors to aid in the evaluation of stored clinical specimens as well as the design and implementation of new pre-clinical evaluations in human subjects. Specifically, we will analyze the immune responses to EtpA and EatA answering the following questions: ? Does the conservation of EtpA and EatA across the spectrum of the ETEC pathovar justify their inclusion in ETEC vaccines? ? How does blood type alter disease outcomes and immune responses to EtpA+ ETEC infections? ? What impact does blood type have on the design and evaluation of clinical vaccine trials? ? What is the best means to evaluate EatA and EtpA immune responses? ? Are aspects of the immune response correlated with protection from infection and if so, can they improve the efficiency of future ETEC vaccine studies? Genomics approaches as well as classic molecular studies will assess the conservation of these antigens while a variety of methods will be used to assess the consequences of each of these antigens on disease outcomes and the immune response based on these antigens. The culmination of these studies will provide data driving the rational design of novel ETEC vaccines as well as the rapid means to assess their efficacy.